The overall goal is to understand the mechanisms by which liver alcohol dehydrogenases (ADH) regulate the metabolism of alcohol. Since ADH catalyzes the rate-limiting step in ethanol metabolism, we propose that the enzymatic properties of ADH play a key role in determining the pharmacokinetics of ethanol elimination. Studies in our laboratory and elsewhere have indicated that there are as many as 8 different genetically determined ADH subunits which combine to form more than 20 different active dimeric isoenzymes. It has been shown that ADH isoenzymes exhibit widely different kinetic properties and recent sequence analysis of ADH subunits and genes has identified some of the substitutions responsible for these differences. Our aim is to systematically examine the function of amino acids in the active site of human liver B1B1 ADH by site-directed mutagenesis. We will create mutagenized forms of the B1 cDNA, which we have recently cloned, by annealing synthetic oligonucleotides containing a mismatched sequence to the cDNA. Mutagenized forms of B1B1 containing the substituted amino acid encoded by the mismatched oligonucleotide will be expressed in yeast. We will examine the steady-state and stopped-flow kinetic properties and substrate specificities of the mutagenized ADHs. By these methods, we will examine structure-function relationships for basic residues which bind the pyrophosphate of coenzyme, residues that participate in the putative alcohol-proton relay system, residues in the alcohol binding pocket, residues which undergo a conformational change upon binding coenzyme, and residues which bind the noncatalytic zinc atom and determine the structure of this site. We will also examine the specificity of formation of complexes of ADH isoenzymes with glyceraldehyde-3-phosphate dehydrogenase and investigate the proposed direct intermolecular transfer of NADH between these dehydrogenases. The study of structure-function relationships in the human liver B1B1 ADH should contribute substantially to the fundamental understanding of the mechanism of catalysis by ADH and to the role that human liver ADH isoenzymes may play in the regulation of alcohol metabolism.